Compositions, methods and kits for biarsenical fluorophore labeling

ABSTRACT

Methods, compositions, and kits for labeling tetracysteine-tagged proteins with biarsenical fluorophores with increased specificity, including compositions, methods and kits particularly adapted for labeling of tetracysteine-tagged proteins to be resolved within an electrophoresis gel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplications Ser. No. 60/514,447, filed Oct. 24, 2003; Ser. No.60/515,011, filed Oct. 27, 2003; and Ser. No. 60/515,575, filed Oct. 28,2003; the disclosures of which are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The invention is drawn to compositions, methods and kits for labelingtetracysteine-tagged proteins with biarsenical fluorophores, withincreased specificity, including compositions, methods and kitsparticularly adapted for labeling of tetracysteine-tagged proteins to beresolved within an electrophoresis gel.

BACKGROUND OF THE INVENTION

Recently, a new class of fluorophore derivatives of tremendous value inthe detection, visualization, and purification of recombinant proteinshas been introduced.

These biarsenical fluorophore derivatives, as adducts with EDT(1,2-ethanedithiol), are self-quenching: the small size of the EDTmoieties permits free rotation of the arsenic atoms, which quenchesfluorescence of the fluorophore. The essentially nonfluorescent moleculeis rendered fluorescent by competitive displacement of the EDT moiety bya specific tetracysteine peptide motif (CCXXCC, where “X” represents anyamino acid), an engineered sequence that is uncommon in naturalproteins; binding to the tetracysteine motif constrains motion of thearsenic atoms, unquenching the fluorophore. Griffin et al., Science281:269 (1998); Griffin et al., Methods Enzymol. 327:565-78 (2000);Adams et al., J. Amer. Chem. Soc. 124:6063-6076 (2002); Gaietta et al.,Science 503-507 (2002); U.S. Pat. Nos. 5,932,474, 6,054,271; 6,451,569;6,008,378; U.S. patent application publication no. 2003/0083373, andinternational patent application publication no. WO 99/21013, thedisclosures of which are incorporated herein by reference in theirentireties.

Advantages of the biarsenical fluorophores as fluorescent proteinlabeling reagents include small size, ability of the EDT₂ adducts tocross cell membranes, ability to recognize a binding domain that issufficiently small as not to interfere substantially with the functionof the protein to which it is fused, nanomolar (or lower) dissociationconstant for binding to the tetracysteine motif, rapid conversion from anonfluorescent to a fluorescent state upon binding, and thereversibility of its binding upon addition of a high concentration(millimolar) of EDT.

The biarsenical derivative of fluorescein that is most commonly used is4′-5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(2,2-ethanedithiol)₂,known as FlAsH™-EDT₂ or Lumio™ Green, and is available commercially(Invitrogen Corp., Carlsbad, Calif.). The red-fluorescing biarsenicalresorufin derivative, known as ReAsH™ or Lumio™ Red, is also availablecommercially; methods of synthesizing other such biarsenicalfluorophores, such as CHoXAsH-EDT₂ and HoXAsH-EDT₂ are described in theliterature.

Tetracysteine biarsenical affinity tags (FlAsH™ tags) have beensuccessfully incorporated at either the N- or C-termini of proteins, aswell as exposed surface regions within a protein and have been used topermit visualization of recombinant proteins expressed within livingcells, and in SDS-PAGE gels. Griffin et al. 1998, Griffin et al. 2000,Adams et al. 2002, supra.

In PAGE gels, inclusion of the FlAsH-EDT₂ reagent in the sample loadingbuffer allows rapid detection of recombinant proteins in whole celllysates using a standard UV light box without the need for westernblotting or other more laborious protein detection methods.

Although the preferred tetracysteine motif occurs rarely in naturalproteins, permitting specific labeling of proteins to which thetetracysteine motif has been recombinantly fused, FlAsH-EDT₂ has beenshown additionally to bind to endogenous cysteine-containing proteins,Stroffekova et al., Pflugers Arch.—Eur. J. Physiol. 442:859-866 (2001),which increases background fluorescence. Stroffekova et al. suggest thatFlAsH™ binding to the vicinal cysteines in the C-X-X-C protein motif ofendogenous proteins may limit the use of FlAsH-EDT₂ to stainingrecombinant proteins expressed at a high level in cells with a naturallylow background.

Given the advantages of biarsenical fluorophores as labeling andpurification reagents for recombinantly tagged proteins, there is a needin the art for compositions, methods, and kits that permittetracysteine-labeled fusion proteins to be labeled with biarsenicalfluorophores with decreased reactivity with endogenous protein motifs.There is a particular need in the art for compositions, methods and kitsthat provide increased specificity of labeling of tetracysteine-taggedrecombinant proteins resolved within electrophoresis gels.

SUMMARY OF THE INVENTION

The present invention solves these and other needs in the art byproviding methods, compositions, and kits for detecting, characterizingand/or purifying tetracysteine-tagged proteins with increasedspecificity.

We have found that we can reduce the spurious binding of biarsenicalfluorophores to vicinal cysteines of endogenous proteins by using mono-and dithiols to compete with the binding reaction; with thecompositions, methods and kits of the present invention, suchcompetition does not substantially hinder the desired binding of thefluorophore to a tetracysteine tag. The compositions, methods and kitsof the present invention are useful in improving specificity ofbiarsenical fluorophore binding in both protein detection methods andprotein purification methods.

We have also found, surprisingly, that the resulting increase inspecificity can be lost during electrophoresis: without intending to bebound by theory, it appears that the pH of certain gels precludesmigration of the uncharged mono- and di-thiols; comigration of thebiarsenical fluor and proteins in the gel thus permits the biarsenicalfluorophores to bind to vicinal thiols that had earlier been blocked bythe mono- and dithiol reagents. For in-gel staining oftetracysteine-tagged recombinant proteins, the invention thus providescompositions and methods for reductive competition in the presence ofthe biarsenical fluor, followed by alkylation, effectively cappingvicinal thiols and preventing binding of the fluor to vicinal thiolsduring electrophoresis, leading to a substantial improvement inspecificity during in-gel staining reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and advantages of the present invention willbe apparent upon consideration of the following detailed description,taken in conjunction with the accompanying drawings, in which likereference characters refer to like parts throughout, and in which:

FIG. 1 shows the structure of FlAsH-EDT₂, a known biarsenicalfluorophore;

FIG. 2 shows the unquenching of FlAsH™ fluorescence upon competitivedisplacement of both EDT moieties by the tetracysteine tag of arecombinantly engineered protein, according to the prior art;

FIG. 3 schematizes the elimination of undesired binding of FlAsH-EDT₂(Lumio™ Green) to vicinal cysteines, with retention of desired bindingof the fluorophore to the engineered tetracysteine motif, by reductionof the proteins in the sample in the presence of a thiol competitor,exemplified by betamercaptoethanol, according to one embodiment of thepresent invention;

FIG. 4 schematizes a further embodiment of the methods of the presentinvention, in which a later incubation with a thiol-reactive blockingagent, such as a thiol-reactive capping agent, is used to preventvicinal thiols from reacting with the biarsenical fluorophore afterelectrophoretic removal of the thiol competitor, exemplified bybetamercaptoethanol, according to one embodiment of the presentinvention;

FIG. 5A shows the fluorescent detection of FlAsH-tagged proteinsexpressed in E. coli cells, with cell lysates treated with thecompositions and according to the methods of the present invention andthen resolved in a 4-12% NuPAGE gel run with MES running buffer; FIG. 5Bshows the same gel after staining with a visible dye;

FIG. 6A shows the fluorescent detection of FlAsH-tagged proteinsexpressed in vitro, with the in vitro reactants treated with thecompositions and according to the methods of the present invention andresolved in a 4-12% NuPAGE gel run with MES running buffer; FIG. 6Bshows the same gel after staining with a visible dye;

FIG. 7A shows the fluorescent detection of FlAsH-tagged proteinsexpressed in a human 293 cell line, with cell lysates treated with thecompositions and according to the methods of the present invention andresolved in a 4-12% NuPAGE gel run with MES running buffer; FIG. 7Bshows the same gel after staining with a visible dye;

FIGS. 8A and 8B show the specificity of labeling of a 48 kDatetracysteine-tagged fusion protein using the reductive competition andsubsequent thiol blocking methods of the present invention, with panel Ashowing fluorescent detection, and panel B showing subsequent visualstaining of the same NuPAGE® Novex 4-12% Bis-Tris gel, of which lane 1was loaded with a spectrally matched fluorescent standard proteinladder, lane 2 loaded with a negative control E. coli extract, and lanes3-5 respectively loaded with 1200 ng, 240 ng, and 48 ng of pure 48 kDaLumio fusion protein mixed with E. coli lysate; and

FIG. 9 shows real-time detection of tetracysteine-tagged proteinproduction, using Lumio™ Green labeling of in vitro synthesizedtetracysteine-tagged protein.

DETAILED DESCRIPTION

In a first aspect, the invention provides methods for labeling one ormore tetracysteine-tagged proteins that are present in an inhomogeneousprotein sample with biarsenical fluorophores; the methods provideincreased specificity for labeling tetracysteine-tagged proteins overlabeling proteins containing vicinal cysteines, thus decreasing spuriousbackground fluorescence. The method comprises: reducing the proteins inthe sample in the presence of a thiol competitor and a biarsenicalfluorophore.

The protein to be labeled can be any protein having a tetracysteinepeptide motif, CCXXCC (SEQ ID NO:1), wherein X is any amino acid.

In preferred embodiments, the tetracysteine tag has the sequence CCPGCC(SEQ ID NO:2). In some embodiments, the sequence Ala-Gly-Gly (AGG) isadded to the N-terminus and Gly-Gly-Gly (GGG) added to the C-terminus ofthe CCPGCC tag to minimize any interference with sequences and motifsbracketing the tetracysteine tag (“tetracysteine tag” and “FlAsH™-tag”are used synonymously herein; “FlAsH™-tag” is not intended to connotethat the tag can be labeled only with FlAsH™ biarsenical fluoresceinderivative) and better to present the tag as an epitope when generatingantibodies against the tag. This 12 amino acid sequence, AGGCCPGCCGGG(SEQ ID NO:3), can be encoded by a nucleotide sequence optimized forexpression in the desired host cell, as further described below, such asthe nucleic acid sequence: 5′-GCT GGT GGC TGT TGT CCT GGC TGT TGC GGTGGC GGC (SEQ ID NO.:4).

Cysteine tags that have fewer or more than 4 cysteines can also be used(e.g., 2, 3, 4, 5 or 6 cysteines). Typically, the intervening Xaa aminoacids have a high propensity to form α-helical structures. A cysteinetag may be arranged such that the side chains of two pairs of cysteinesare exposed on the same face of an α-helix. A cysteine tag need not becompletely helical to react with a biarsenical reagent. For example, andwithout intending to be bound by theory, reaction of a first arsenic ofa biarsenical with a pair of cysteines may nucleate an α-helix andposition two other cysteines favorably for reacting with a biarsenicalmolecule.

The tetracysteine peptide tag is typically recombinantly fused to theprotein desired to be labeled, either at the N-terminus, C-terminus, orin frame within the protein sequence; expression vectors for creatingtetracysteine-fused recombinant proteins may readily be constructedusing art-routine procedures.

In addition to the tetracysteine tag, other protein sequences canusefully be recombinantly appended to the proteins desired to belabeled. Among such additional protein sequences are linkers—so as tospace the tetracysteine tag at a sufficient distance from criticalresidues of the protein to be labeled as to permit its proper folding,either for retention of biological function or immunogenicity, orboth—and other short tags, usefully epitope tags, such as a FLAG tag, ora myc tag. Other sequences usefully included in the protein fusioninclude short tags useful for purification, such as a polyhistidine(e.g., 6xhis) tag.

In other embodiments, the tetracysteine tag is chemically conjugated tothe protein to be labeled using art-routine conjugation chemistries.

The protein desired to be labeled can include a single tetracysteine tagor a plurality of tetracysteine tags; in embodiments in which theprotein includes a plurality of recombinantly fused tags, the tags maybe separated from one another within the primary amino acid sequence ofthe protein or directly multimerized in tandem.

The protein to which the tetracysteine tag is fused or conjugated can beany protein desired to be labeled, either naturally-occurring ornonnaturally occurring. Naturally-occurring proteins may have knownbiological function or not, and may be known to be expressed or onlypredicted from genomic sequence. The protein, if naturally-occurring,can be a complete protein or only a fragment thereof.

The tetracysteine-tagged protein desired to be labeled can thus be ananimal protein, such as a human protein or non-human mammalian protein,a fungal protein, a bacterial protein, including eubacterial andarchaebacterial protein, a plant protein, an insect protein or a viralprotein.

The sample can be any protein sample in which the tetracysteine-taggedprotein is present.

In one series of embodiments, for example, the tetracysteine-taggedprotein is expressed recombinantly in host cells. In such embodiments,the sample is typically a lysate of the host cells. The lysate can beunpurified, partially purified, or substantially purified.

For example, the host cells can be bacterial host cells. Suitablebacterial host cells include gram negative and gram positive bacteria ofany genus, including Escherichia sp. (e.g., E. coli), Klebsiella sp.,Streptomyces sp., Streptococcus sp., Shigella sp., Staphylococcus sp.,Erwinia sp., Klebsiella sp., Bacillus sp. (e.g., B. cereus, B. subtilisand B. megaterium), Serratia sp., Pseudomonas sp. (e.g., P. aeruginosaand P. syringae) and Salmonella sp. (e.g., S. typhi and S. typhimurium).Suitable bacterial strains and serotypes suitable for the invention caninclude E. coli serotypes K, B, C, and W. A typical bacterial host is E.coli strain K-12.

Host cells can be fungal host cells (such as Saccharomyces cerevisiaecells), insect cells, plant cells, or mammalian cells (including humancells).

In other embodiments, the tetracysteine-tagged protein is expressed invitro, in which case the sample is the cell-free extract in whichtranslation (and, optionally, transcription) is performed, or apartially purified or purified fraction thereof. In embodiments in whichthe extract permits coupled transcription and translation in a singlecell-free extract, such as the E. coli-based ExpressWay or ExpresswayPlus systems (Invitrogen Corp., Carlsbad, Calif.), the sample is thecell-free extract in which transcription and translation commonly occur,or a fraction thereof.

Usefully, such in vitro transcription/translation extracts lack the SlyDpolypeptide, which has been shown to interact with biarsenical reagents;in some embodiments, such extracts are prepared from host strainsengineered to lack the SlyD polypeptide, such as the following exemplarystrains deposited in the Agricultural Research Service Patent CultureCollection maintained by the National Center for AgriculturalUtilization Research in Peoria, Ill., USA, as shown in Table 1. TABLE 1Accession Strain Genotype No. JDP670 F⁻ ompT hsdS_(B) (r_(B) ⁻m_(B) ⁻)gal dcm B-30688 slyD::kan (DE3) JDP671 F⁻ araD139 delta (argF-lac) U169B-30689 prsL150 relA1 deoC1 rbsR fthD5301 fruA25 slyD1 Tn10 (Tet⁻R)lambda- JDP687 Hfr rna-19 gdhA2 his-95 relA1 B-30690 spoT1 metB1 slyD1Tn10 (Tet⁻R) JDP689 Hfr rna-19 gdhA2 his-95 relA1 B-30691 spoT1 metB1slyD::kan JDP694 F⁻ ompT hsdS_(B) (r_(B) ⁻m_(B) ⁻) gal dcm B-30692 slyD1(DE3) JDP704 F⁻ ompT hsdS_(B) (r_(B) ⁻m_(B) ⁻) gal dcm B-30693 rne131slyD1 (DE3) JDP707 F⁻ ompT hsdS_(B) (r_(B) ⁻m_(B) ⁻) gal dcm B-30694rne131 slyD::kan (DE3)

In vitro transcription/translation extracts derived from such slyDmutant strains are further described in co-pending and commonly-ownedU.S. patent application Ser. No. 10/954,951 (Hanson et al.,“Compositions and Methods for Synthesizing, Purifying and DetectingBiomolecules,” filed Oct. 1, 2004); and co-pending and commonly-ownedU.S. provisional application No. 60/508,142 (Hanson, “Compositions andMethods for Purifying and Detecting Biomolecules,” filed Oct. 1, 2003),the disclosures of which are incorporated herein by reference in theirentireties. Other in vitro transcription/translation systems suitablefor use with the present invention are further described in co-pendingand commonly-owned U.S. provisional application No. 60/614,590 (Kudlickiet al., “Feeding Buffers, Systems, and Methods for In Vitro Synthesis ofBiomolecules,” filed Oct. 1, 2004), the disclosure of which isincorporated herein by reference in its entirety.

Moreover, proteins can be tagged using a motif taken from or based onthe slyD sequence, such that the tagged protein can bind biarsenicalfluorophores. Such slyD-tagged proteins can also be used with thepresent invention to achieve labeling with increased specificity. SuchslyD-based tags are further described in co-pending and commonly-ownedU.S. patent application entitled “Target Sequences for SyntheticMolecules” (Hanson, filed Oct. 22, 2004, Atty. Docket No. 0942.6590001)the disclosure of which is incorporated herein by reference in itsentirety.

Such slyD strains are not required, however, since the methods,compositions, and kits of the present invention improve specificity oflabeling of tetracysteine-tagged proteins even in the presence of SlyDand similar cysteine-rich proteins.

In the methods of the present invention, the proteins in the sample aretreated in the presence of a composition comprising thiol competitor anda biarsenical fluorophore. The thiol competitor may serve additionallyas a reducing agent. In some embodiments of the present invention, thecomposition may further comprise an additional reducing agent inaddition to the thiol competitor.

Reducing agents usefully include dithiothreitol (DTT),tris(2-carboxyethyl)phosphine (TCEP), tri-n-butylphosphine (TBP),2-mercaptoethanol (2-ME or β-ME), and mercaptoethanesulfonic acid (MES),and combinations thereof. Other reducing agents may also be used.

The reducing agents are usefully present in concentrations of at leastabout 10 μM, typically at least about 100 μM, 1 mM, 2.5 mM, 3.75 mM, 5mM, 10 mM, 50 mM, 100 mM, even at least about 350 mM, and inconcentrations typically no more than about 500 mM, more typically nomore than about 350 mM, 200 mM, 100 mM, often no more than about 50 mM,10 mM, 5 mM, 2.5 mM, even on occasion no more than about 100 μM or even10 μM.

Thiol competitors, as defined herein, are compounds that are useful forcompeting with other thiol functional groups, such as those found invicinal cysteines, for binding to biarsenical fluorophores. In someembodiments, the thiol competitors usefully may have a higher, similar,or lower affinity with the biarsenical fluorophores than thenon-specific thiol groups, such as those found in slyD. In someembodiments, the thiol competitor may have a lower affinity to thebiarsenical fluorophore than the specific tetracysteine tag describedherein.

Thiol competitors usefully include 2-mercaptoethanol (2-ME or β-ME),1-mercaptopropanol, 2-mercaptopropanol, 1- mercaptobutanol,2-mercaptobutanol, mercaptoethanesulfonic acid (MES),1-mercapto-1,2-propanediol (MPD), mercaptoacetic acid, mercaptosuccinicacid, 3-mercaptopropionic acid, cysteine, cys-cys dipeptide,2,3-dimercapto-1-propanesulfonic acid (DMPS),meso-2,3-dimercaptosuccinic acid (DMS), 2,3-dimercapto-1-propanol (DMP),benzenethiol, and 4-methylbenzenethiol. Among mono- and di-thiolcompetitors, at present monothiols are preferred to dithiols. Otherthiol competitors may also be used. It is understood that the foregoingthiol competitors may also act as reducing agents.

Thiol competitors (equivalently, “thiol binding competitors” or“competitors”) are usefully present in concentrations of at least about10 μM, typically at least about 100 μM, 1 mM, 2.5 mM, 5 mM, 10 mM, 50mM, 100 mM, even at least about 350 mM, 400 mM, even at least about 450mM or 500 mM, and in concentrations typically no more than about 500 mM,more typically no more than about 350 mM, 200 mM, 100 mM, often no morethan about 50 mM, 10 mM, 5 mM, 2.5 mM, even on occasion no more thanabout 100 μM or even 10 μM.

In preferred embodiments, the thiol competitor is itself a reducingagent.

In one such embodiment, the thiol competitor is β-ME, usefully in aconcentration of greater than about 1 mM, often at a concentration ofgreater than about 2 mM, 3 mM, 4 mM, even greater than about 5 mM. Insome embodiments, β-ME is present at a concentration of greater thanabout 10 mM, 20 mM, 30 mM, and may be present in a concentration of 40mM, 50 mM, 60 mM, 65 mM, 70 mM, even 100 mM, 200 mM, 300 mM or higher,with intermediate values permissible. In some embodiments, the thiolcompetitor is β-ME at a concentration of no more than about 500 mM,typically no more than about 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200mM, 150 mM, even no more than about 100 mM.

In another series of embodiments, the reducing agents are TBP and β-ME,with β-ME serving additionally as a thiol competitor. In these latterembodiments, the TBP can usefully be present in a concentration of atleast about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, even at least about 10 mM, andtypically less than about 10 mM, 5 mM, and often at about 3-5 mM, withintermediate values, such as 4 mM, permissible and useful.

In the presence of TBP, β-ME can usefully be present at a concentrationof at least about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, even at leastabout 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, even 70 mM ormore, and typically at a concentration of less than about 100 mM, 95 mM,90 mM, 85 mM, 80 mM, with intermediate values permissible and aconcentration of about 70 mM proving useful.

In some embodiments, the thiol competitor is a monothiol, usefully at aconcentration greater than about 5 mM, often at a concentration greaterthan about 20 mM, 50 mM, even greater than about 100 mM. In otherembodiments, the thiol competitor is a dithiol, usefully at aconcentration of about 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, even atleast about 1 mM, 1.5 mM, 1.75 mM, 2 mM, 5 mM, or 10 mM or higher.

In the methods of the present invention, the biarsenical fluorophore canusefully be a biarsenical derivative of a known fluorophore, such asfluorescein, usefully FlAsH-EDT₂ (Lumio™ Green, Invitrogen Corp.,Carlsbad, Calif.), or such as resorufin, usefully ReAsh-EDT₂ (Lumio™Red, Invitrogen Corp., Carlsbad, Calif.), or may instead be an oxidizedderivative, such as ChoXAsH-EDT₂ or HoXAsH-EDT₂.

The biarsenical fluorophore can be a biarsenical derivative of otherknown fluorophores, including, e.g., the Alexa fluor series, asdescribed in U.S. Pat. No. 6,130,101, incorporated herein by referencein its entirety, including Alexa Fluor-350, Alexa Fluor-430, AlexaFluor-488, Alexa Fluor-532, Alexa Fluor-546, Alexa Fluor-568, AlexaFluor-594, Alexa Fluor-663 and Alexa Fluor-660, available commerciallyfrom Molecular Probes (Eugene, Oreg.).

The biarsenical fluorophore can be any of the biarsenical fluorophoresdescribed in U.S. Pat. No. 6,054,271, incorporated herein by referencein its entirety.

The biarsenical fluorophore can be present at a concentration of atleast about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50μM, 100 μM or more, and at a concentration of no more than about 500 μM,400 μM, 300 μM, 200 μM, 100 μM, 90 μM, 80 μM, 70 μM, even no more thanabout 60 μM, 50 μM, or even no more than about 40 μM, with intermediatevalues permissible. For FlAsH-EDT₂(4′-5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(2,2-ethanedithiol)₂),the concentration can usefully be at least about 5 μM, 10 μM, 20 μM, 30μM, 40 μM, 50 μM, often no more than about 100 μM, 90 μM, 80 μM, 70 μM,60 μM, 50μM, 40 μM, with intermediate values permissible and useful, andwith a concentration of about 10 μM being particularly useful.

The order of addition of reducing agent, thiol competitor, andbiarsenical fluorophore is not critical.

Typically, the reducing agent and thiol competitor are added to theprotein sample prior to addition of biarsenical fluorophore. In someembodiments, the reducing agent and thiol competitor are separate agentscommonly included in a single fluid composition, and are thus addedsimultaneously to the protein sample. In other embodiments, the reducingagent and thiol competitor are present as separate compounds (or, ifliquid, neat) or in separate compositions, and are added separately, ineither order, to the protein sample. In other embodiments, the reducingagent and the thiol competitor are the same.

In yet other embodiments, the biarsenical fluorophore is added before,or concurrently with, one or both of the reducing agent and thiolcompetitor.

Optionally, treatment of the proteins in the sample by incubation in thepresence of a thiol competitor (and, optionally, also the biarsenicalfluorophore) is carried out at temperatures above room temperature, suchas at a temperature of at least about 30° C., 40° C., 50° C., even atleast about 60° C., 70° C., or 80° C., even as high as 90° C., 95° C.,96° C., 97° C. even as high as 100° C., with intermediate valuespermissible, and typically at a temperature no more than about 97° C.,96° C., 95° C., 90° C., 80° C., even no more than about 70° C., withintermediate values permissible. As further described in Example 1,below, a temperature of 70° C. usefully accelerates the reductivelabeling, in the presence of a thiol competitor, of thetetracysteine-tagged protein desired to be labeled.

Incubation can also be performed at room temperature. In suchembodiments, the incubation period is usefully increased. The durationof incubation can readily be determined by observing the increase influorescence over time, and determining the timepoint beyond whichfluorescence does not increase.

Additional agents may usefully be added to the protein sample,including: density-adjusting agents, such as sugars and polysaccharides,including glycerol or sucrose; buffering agents, such as Tris base andTris HCl; detergents, such as sodium dodecyl sulfate (SDS) or lithiumdodecylsulfate (LDS); chelating agents, such as EDTA or EGTA; andvisually detectable dyes, such as Serva Blue G or bromophenol blue, withsuch visually detectable dyes typically chosen so as not to absorbsignificantly at the emission maximum of the biarsenical fluorophore.

The sample proteins are reductively incubated with the thiol competitorand biarsenical fluorophore for at least 30 seconds, typically at least1 minute, more typically at least 2 mins, 3 mins, 4 mins, 5 mins, even6, 7, 8, 9, 10 minutes, with times of at least 20 mins, 30 mins, 40mins, 50 mins, even at least 1 hour, and times intermediate thereto,permissible. In some embodiments, the sample proteins can be reductivelyincubated with the thiol competitor and biarsenical fluorophore for atleast about 2 hours, 3 hours, even at least about 4 hours or more.

Typically, the sample proteins are reductively incubated (i.e.,incubated in the presence of a reducing agent) in the presence of athiol competitor and biarsenical fluorophore for fewer than about 5hours, 4 hours, 3 hours, 2 hours, even less than 1 hour, particularlywhen incubation is optionally performed at temperatures above roomtemperature, as described below. Times intermediate thereto are alsopermissible.

The duration of reductive incubation in the presence of a thiolcompetitor and biarsenical fluorophore is typically not critical, andmay be conveniently adjusted to the needs of the experiment.

As shown in the exemplary methods of Example 1, reductive incubation inthe presence of a thiol competitor and biarsenical fluorophore canusefully be performed at 70° C. in as few as 10 minutes. As furthershown in Example 1, the protein sample can thereafter be allowed tocool, typically to room temperature, when reductive incubation in thepresence of a thiol competitor and biarsenical fluorophore is performedat elevated temperature.

In some embodiments of the methods of the present invention, a controlprotein may usefully be labeled to monitor the effectiveness ofbiarsenical fluorophore labeling, either in a parallel reaction or, ifreadily resolvable from the protein desired to be labeled, by inclusionin the same reaction.

In one such embodiment, particularly useful as a control for labelingwith a biarsenical derivative of fluorescein, such as FlAsH™ (Lumio™Green, Invitrogen Corp., Carlsbad, Calif.), the control protein canusefully be tetracysteine-tagged cyan fluorescent protein (CFP),effective labeling of which can be monitored as a decrease influorescence emission at 485 nm upon excitation at about 430 nm, theemission maximum of native CFP, with concomitant increase in emission at535 nm due to fluorescence resonance energy transfer between the CFP andcovalently bound FlAsH™ molecule.

The methods of the present invention significantly reduce the spuriousbinding of the biarsenical fluorophore to proteins lacking thetetracysteine tag without significantly reducing the binding of thebiarsenical fluorophore to proteins having the tetracysteine tag,substantially increasing specificity of binding of biarsenicalfluorophores to tetracysteine tags.

As shown in FIGS. 5A-5B, 6A-6B, 7A-7B, and 8A-8B, and furtherdemonstrated in the Examples below, the methods of the present inventionmay readily be used to label, with high specificity, a wide range oftetracysteine-tagged proteins that are expressed in E. coli, that areexpressed in mammalian cells, and that are expressed in vitro in acoupled transcription-translation cell-free extract, with high signaland high specificity.

As also shown in FIGS. 5-8, the methods of the present invention findparticular use in labeling tetracysteine-tagged proteins that aresubsequently to be resolved by polyacrylamide gel (PAGE)electrophoresis, permitting the in-gel detection of tetracysteine-taggedproteins without gel drying, without blotting, and without expensive andcomplex equipment.

Thus, in another series of embodiments, the methods of the presentinvention can usefully include a subsequent step of resolving the sampleproteins by electrophoresis, such as gel electrophoresis, capillaryelectrophoresis, or fluid (solution) phase electrophoresis.

For size fractionation, the labeled proteins can be resolved, forexample, by gel electrophoresis, for example by electrophoresis in a 10%Bis-Tris gel, a 4-12% Bis-Tris gel, or a 12% Bis-Tris Gel, such as aNuPAGE® Novex Bis-Tris gel (Invitrogen Corp., Carlsbad, Calif.) run witheither MES or MOPS buffer. In other such embodiments, the proteins canbe resolved in a Tris-acetate (TA) gel, such as a 7% or 3-8% gradientTris-acetate gel, such as a NuPAGE® Novex Tris acetate gel (InvitrogenCorp., Carlsbad, Calif.). In yet other embodiments, the proteins can beresolved in a Tris-glycine (TG) gel, including 4% TG gels, 6% TG gels,8% TG gels, 10% TG gels, 12% TG gels, 14% TG gels, 16% TG gels, and 18 %TG gels, and TG gels having gradients such as 4-12%, 4-20%, 8-16%,10-20%, such as the TG gels available from Invitrogen Corp. (Carlsbad,Calif.). In yet other embodiments, the proteins can be resolved inTricine gels, such as 10% Tricine gels, 16% Tricine gels, or even 10-20%gradient Tricine gels, and in standard Laemmli gels.

The gel-based electrophoretic embodiments of the methods of the presentinvention can be carried out in gels of any suitable physical format,for example in standard-sized gels, minigels, strips, in gels designedfor use with microtiter plates and in other high throughput (HTP)applications.

For example, up to 96 protein samples labeled according to the methodsof the present invention can be resolved simultaneously in an E-PAGE™High-Throughput (HTP) Protein Electrophoresis System (Invitrogen Corp.,Carlsbad, Calif.). The E-PAGE™96 gels are self-contained, pre-cast gelsthat include a gel matrix and electrodes packaged inside a disposable,UV-transparent cassette. Each E-PAGE™ 96 gel contains 96 sample lanesand 8 marker lanes in a staggered well format. The well openings of theE-PAGE™ 96 cassette are compatible with the standard 96-well plateformat and can be conveniently loaded with a multichannel pipettor or8-, 12-, or 96-tip liquid-handling robotic devices. In addition, eachE-PAGE™ 96 cassette is labeled with an individual barcode to facilitateidentification of the gel using commercial barcode readers. The E-PAGE™gel matrix is further described in commonly owned and copendingprovisional patent application Nos. 60/504,683, filed Sep. 19, 2003;60/508,786, filed Oct. 2, 2003; 60/560,310, filed Apr. 6, 2004; and U.S.patent application Ser. No. 10/946,472 (Updyke et al., “CompositeCompositions for Electrophoresis,” filed Sep. 20, 2004), the disclosuresof which are incorporated herein by reference in their entireties.

Gel formats within which the labeled proteins of the present inventionmay usefully be resolved include without limitation those described inthe following patents and published patent applications: U.S. Pat. Nos.5,578,180; 5,922,185; 6,059,948; 6,562,213; 6,057,106; 6,096,182;6,143,154; 6,162,338; U.S. Patent application publication nos.2002/0134680 A1; 2003/0127330 A1; and 2003/0121784 A1; and published PCTApplication Nos. WO 95/27197, WO 99/37813, WO 02/18901, WO 02/071024,the disclosures of which are incorporated herein by reference in theirentireties.

The embodiments of the present invention that include electrophoreticseparation of biarsenically labeled proteins may also be practiced usingcapillary electrophoresis (CE) or capillary zone electrophoresis (CZE).

For fractionation based on isoelectric point, the proteins can beresolved using isoelectric focusing (IEF), either as a single separationstep or as a step preliminary to size fractionation (i.e., as the firststep in 2D-PAGE).

For example, the labeled proteins can be resolved using solution phaseisoelectric focusing. Zuo et al., Anal. Biochem. 284: 266-278 (2000);Zuo et. al., Electrophoresis 22: 1603-1615 (2001); Zuo et al.,Proteomics: 2: 58-68 (2002); Zuo et al., Journal of Chromatography B782: 253-265 (2002); Ali-Khan et al., Current Protocols in ProteinScience 22.1: 1-19 (2002), the disclosures of which are incorporatedherein by reference in their entireties. Devices and kits for fluidphase isoelectric focusing are available commercially (ZOOM® IEFFractionator, Invitrogen Corp., Carlsbad, Calif.). In such embodiments,the proteins can be labeled with biarsenical fluorophore before or afterfractionation with solution phase IEF.

In other embodiments, the labeled proteins can be resolved usinggel-based isoelectric focusing, either in slab gels, in tube gels, or,in one series of embodiments, in immobilized pH gradient (IPG) strips.

IPG strips are available commercially (Zoom® IPG strips, InvitrogenCorp., Carlsbad, Calif.). Devices that substantially facilitate theirusage have recently been described—e.g., in commonly owned internationalpatent application published as WO 02/092200, concurrently pending andcommonly owned U.S. Pat. Appl. Publ. No. 2003/0015426, and concurrentlypending and commonly owned U.S. patent application Ser. No. 10/464,258,filed Jun. 17, 2003, the disclosures of which are incorporated herein byreference in their entireties. Such devices are available commercially(Zoom® IPGRunner system, Invitrogen Corp., Carlsbad, Calif.).

Although the methods above-described for labeling tetracysteine-taggedproteins can usefully be followed, in a wide variety of embodiments, bya subsequent step of electrophoresis, we have found that the resultingincrease in labeling specificity using the methods of the presentinvention can surprisingly be lost during some types of gelelectrophoresis, particularly polyacrylamide gel electrophoresis inneutral pH gels.

Without intending to be bound by theory, it appears that the neutral pHof certain gels—such as the long shelf life, prior-cast, high resolutionNuPAGE™ bis-tris gels—precludes significant migration into the gel ofuncharged mono- and di-thiol thiol competitors; comigration of thebiarsenical fluor and sample proteins in the gel in the absence of suchthiol competitors permits the biarsenical fluorophores to bind tovicinal thiols that had earlier been blocked by the thiol competitors.

Accordingly, in some embodiments of the methods of the presentinvention, a blocking agent may usefully be added after the step ofreductive incubation with thiol competitor and biarsenical fluorophore,and before further analytical or preparative steps, such as resolutionby electrophoresis. Without intending to be bound by theory, it isbelieved that the blocking agent caps vicinal thiols and preventsbinding of the fluor to those thiols during electrophoresis.

The blocking (equivalently herein, “enhancing”) agent may usefully be anagent capable of alkylating, or otherwise covalently capping, freesulfhydryls.

The blocking agent can thus be an alkylating agent, such as anacylhalide or alkylhalide, such as a haloacetamide, such asiodoacetamide or iodoacetic acid; a dithiol that will form a stabledisulfide bond with sulfhydryls, such as dithiobis(2-nitrobenzoic acid)(DTNB), dithiobis(5-nitropyridine); or a maleimide, such as maleimide orethylenemaleimide. Other blocking agents include Ellman's reagent andmethyl triflate.

Other useful blocking agents include, for example, 4-vinylpyridine,acrylamide, dimethylacrylamide, and others.

One disadvantage of iodoacetamide as a blocking agent in the methods ofthe present invention is that it is difficult to solubilize at highconcentrations in water. Accordingly, in embodiments in whichiodoacetamide is used as the blocking reagent, it is preferablysolubilized in other solvents, such as dimethylformamide,dimethylsulfoxide, isopropanol, acetonitrile, ethanol, and methanol,with methanol currently preferred for high concentration stocksolutions.

The sample is typically incubated in the presence of blocking agent forat least 1 minute, 2 mins, 3 mins, 4 mins, even at least 5 mins, withincubation times of 10 mins, 20 mins, even 30 mins, 60 mins, or 2 hrs ormore possible, with times intermediate thereto permissible. Incubationin the presence of blocking reagent can usefully be fewer than 60 mins,50 mins, 40 mins, even fewer than 30 mins. As shown in the embodimentsof Example 1, an incubation of no more than 5 minutes is useful.

Typically, incubation in the presence of blocking reagent is performedat room temperature, although incubation at elevated or reducedtemperature also be performed.

Surprisingly, a lower concentration of blocking agent than would beexpected to be required, based upon the concentration of concurrentlypresent thiol competitor, can be used effectively. Thus, if the thiolcompetitor is βME at a concentration of 100 mM, 90 mM, 80 mM, even 70mM, effective concentrations of blocking reagent, such as maleimide,DTNB or iodoacetamide, can be as low as 100 mM, 75 mM, 50 mM, 40 mM,even as low as 25 mM.

Addition of the blocking reagent leads to a substantial improvement inspecificity of labeling of tetracysteine-tagged proteins in neutral pHgels.

In embodiments in which the labeled proteins are resolved byelectrophoresis, the methods may usefully include a further step ofvisualizing the labeled proteins.

Such visualization can usefully be done with the proteins still presentin an intact gel (in-gel detection), by exciting the fluorophore using aUV transilluminator or a laser-based scanner.

For example, in embodiments in which the biarsenical fluorophore is afluorescein derivative, such as Lumio™ Green (FlAsH™), the gel can beplaced on a UV transilluminator equipped with a standard camera havingeither an ethidium bromide or SYBR™ Green-appropriate filter.Alternatively, a laser-based scanner can be used, such as the Typhoon™or Storm™ Scanners (Amersham Biosciences, Piscataway, N.J.), using alaser line that falls within the excitation maximum of the fluorophore(500 nm), and a 535 nm long pass filter or band pass filtered centerednear the emission maximum of 535 nm.

Optionally, the gel is first removed from the cassette; depending uponthe UV-attenuating characteristics of the cassette materials, removal ofthe gel can increase sensitivity of detection.

Using the methods and compositions of the present invention, proteinscan be detected in electrophoresis gels with high sensitivity. Themethods and compositions permit detection of no more than about 1 pMoltetracysteine-tagged protein, more typically detection of no more thanabout 5 pMol protein, and can detect 10 pMol, 25 pMol or more. Themethods and compositions of the present invention can detect as littleas 1 ng of tagged protein, 2 ng, 3 ng, 4 ng, even as little as 5 ngprotein.

In one series of embodiments, the proteins of the labeled sample canusefully be resolved in parallel with a series of fluorescent molecularweight standards.

Usefully, the standards are spectrally matched to the biarsenicalfluorophore used to label the proteins. Such spectral matching can beaccomplished, for example, by using tetracysteine-tagged proteinstandards that are labeled in parallel with the same biarsenicalfluorophore used to label the protein sample, or by using standardshaving a fluorescent moiety that is spectrally matched to thebiarsenical fluorophore used to label the sample proteins. Examples ofstandards useful in the practice of the present invention include theBenchmark™ family of protein standards (Invitrogen Corp., Carlsbad,Calif.).

Such spectral matching permits the standards to be excited at awavelength that commonly excites the biarsenical fluorophore, andfurther permits the fluorescence emission of the standards to becommonly detected with that of the biarsenically-labeled sampleproteins.

In some embodiments, the tetracysteine-tagged protein can be eluted fromthe gel following in-gel visualization of the biarsenical fluorophoreand then further analyzed or purified. In some of these latterembodiments, the biarsenical fluorophore is released from thetetracysteine-tagged protein, typically using 1,2-ethanedithiol (EDT),dithiothreitol (DTT), or 2,3-dimercaptopropanesulfonate (DMPS).

The methods, compositions and kits of the present invention can also beused to increase specificity of binding by biarsenical fluorophores totetracysteine-tagged proteins for purposes other than gelelectrophoresis.

For example, the methods, compositions, and kits of the presentinvention may be used to increase the specificity of tetracysteinelabeling for quantitation of yield of in vitro synthesized protein. Insuch embodiments, aliquots can be withdrawn periodically from the invitro translation (in particularly useful embodiments, from the coupledtranscription-translation) reaction, the translation reaction stopped,and the sample then reduced in the presence of a thiol competitor andbiarsenical fluorophore, and optionally thereafter with thiol-blockingreagent, according to the methods of the present invention. In asubsequent step, the amount of fluorescence is quantitated, as byfluorometry.

In in vitro translation (and, in particularly useful embodiments, incoupled transcription-translation) systems that tolerate the presence ofthiol competitor at concentrations effective in the methods of thepresent invention, monitoring of tetracysteine-labeled protein synthesiscan be performed in real-time, without taking aliquots. In such case,the duration of incubation in the presence of reducing agent, thiolcompetitor, and biarsenical fluorophore vary continuously as well.

An example of such real-time measurement, albeit without addition of thethiol competitor or blocking reagents according to the methods of thepresent invention, is presented in Example 4 and FIG. 9.

Real-time monitoring of protein expression usefully enables confirmationof protein expression without gels, evaluation oftranscription/translation regulators, screens for expression. Moreover,these and other real-time monitoring methods may be performed in a highthroughput manner.

As another example, the methods of the present invention can increasethe specificity of purification of tetracysteine-tagged proteins usingbiarsenical affinity supports, such as the FlAsH™ affinity resindescribed in Thorn et al., “A novel method of affinity-purifyingproteins using a bis-arsenical fluorescein,” Protein Sci. 9:213-217(2002), the disclosure of which is incorporated herein by reference inits entirety. In such embodiments, the biarsenical compound does notneed to be a biarsenical derivative of a fluorophore; it can be anybiarsenical with spacing suitable for binding to a tetracysteine motif.

In a typical embodiment, a protein sample having a tetracysteine-taggedprotein desired to be purified is reductively incubated with a thiolcompetitor and with a biarsenical moiety (either with or without afluorophore) that is attached, often covalently, to a support.

The support may include one or more surfaces of a unitary object, suchas a slide, dipstick, microtiter plate well, MALDI source, or SELDIsource, or may instead comprise the surfaces of a plurality of discreteobjects, such as beads.

The support may be glass, although other solid materials, such as metal,amorphous silicon, crystalline silicon, or plastics, may also be used.Such plastics include polymethylacrylic, polyethylene, polypropylene,polyacrylate, polymethylmethacrylate, polyvinylchloride,polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal,polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, andmixtures and copolymers thereof.

The support may be a surface of a bead, or pellet. The beads may be ofsubstantially spherical geometry, but need not be limited to such. Inaddition, the beads may be porous or otherwise permit access to itsinterior spaces and surfaces, thus increasing the available surface areaof the bead available for purification. Bead dimensions usefully rangefrom nanometers, e.g. 100 nm, to millimeters, e.g. 5 mm, usefully fromabout 0.2 micron to about 200 microns, with beads having dimensions fromabout 0.5 to about 5 microns being typical.

Suitable bead compositions include polymers often used in affinitychromatography, such as agarose beads, or controlled pore glass,plastics, such as polystyrene, methylstyrene, acrylic polymers,ceramics, glass, paramagnetic materials, titanium dioxide, latex,cross-linked dextrans, cellulose, and nylon. See, e.g., “MicrosphereDetection Guide” (Bangs Laboratories, Inc.).

Usefully, the beads are magnetic, paramagnetic, or superparamagnetic,permitting separation of the beads from the liquid sample by applicationof a suitable magnetic field. A variety of such beads may be purchasedcommercially from Dynal® Biotech Inc. (Lake Success, N.Y.) and MiltenyiBiotec Inc. (Auburn, Calif.), and biarsenical fluorophores conjugatedthereto using standard chemistries, such as that described in Thorn etal., “A novel method of affinity-purifying proteins using abis-arsenical fluorescein,” Protein Sci. 9:213-217 (2002).

In some embodiments, the support comprises a plurality of beads,optionally magnetic, paramagnetic, or superparamagnetic, the beadsfurther comprising a bead-purification moiety. The bead-purificationmoiety may, for example, be one member of a high affinity binding pair,such as biotin or streptavidin, permitting the beads (andtetracysteine-tagged protein bound thereto) subsequently to be purifiedusing the other member of the binding pair.

In some embodiments, the purification methods include a further step ofadding a blocking (enhancing) agent, as described herein, after the stepof reductive incubation with thiol competitor and support-boundbiarsenical fluorophore.

In yet other embodiments, the support comprises a gel or resin, and thebiarsenical fluorophore-bound gel or resin is retained within a column,usefully a column having chemicophysical properties that ensure lownonspecific protein adsorption to the column itself.

In such embodiments, the reducing agent, thiol competitor, biarsenicalfluorophore, and optional thiol blocking agent may usefully be selectedfrom those described above. Analogously, incubation times andconcentrations of reducing agent, thiol competitor, biarsenicalfluorophore, and optional thiol blocking agent are usefullysubstantially similar to those described above.

In some embodiments, the thiol competitor is added to the sample priorto contact of the sample to the biarsenical affinity resin or gel. Inother embodiments, the thiol competitor is included in the columnequilibration buffer. In yet other embodiments, the thiol competitor isincluded both in the sample prior to contact of the sample to theaffinity resin or gel and is additionally included in the columnequilibration buffer.

The purification methods typically further comprise a step of removingthe support from the protein sample, or equivalently, of removing theprotein sample from the support, as by washing the solid support toremove nonspecifically bound proteins.

The purification methods may further comprise a subsequent step ofreleasing the tetracysteine-tagged protein from the biarsenicalfluorophore support. Typically, release is effected using1,2-ethanedithiol (EDT), dithiothreitol (DTT),2,3-dimercaptopropanesulfonate (DMPS).

Purification of tetracysteine-tagged proteins using the methods of thepresent invention can achieve protein purities of at least about 70%,75%, 80%, 85%, 90%, 95%, even 96%, 97%, 98% or even more by weight.

The methods and compositions of the present invention can also be usedto quantitate the amount of tetracysteine-tagged protein present in asample.

In such embodiments, the methods of the present invention furthercomprise quantitating the amount of fluorescence from the biarsenicalfluorophore.

The quantitation can be done without resolution of the proteins presentin the protein sample or after the proteins have been partially or fullyresolved, as by electrophoresis, such as PAGE, 2D-PAGE, or IEF.Fluorescence can be quantitated by, e.g., a fluorometer. Quantitation ofthe proteins can also be performed by absorption spectrometry.

In another aspect, the invention provides compositions useful in thepractice of the methods of the present invention.

In one embodiment, the invention provides a composition comprising areducing agent and a thiol competitor at concentrations useful in thepractice of the methods of the present invention. In another embodiment,the composition further comprises a biarsenical fluorophore at aconcentration useful in the practice of the methods of the presentinvention.

In some embodiments of the invention, the biarsenical fluorophore isprepared as a stable formulation. That is, the formulation can be storedat −80° C., −20°C., 4°C., ambient temperature or even highertemperatures for a period of time with little or no loss of function.Preferably, such formulations are stable for a period of time such as 2years, 12 months, 6 months, 3 months, 60 days, 30 days or 2 weeks. Apreferred stable formulation is one that may be used directly as aloading solution or a gel-staining solution, or as a stock solution thatcan be diluted in order to generate such solutions.

In yet another aspect, the invention provides a kit for use in thepractice of the methods of the present invention.

In one series of embodiments, the kit provides a first compositioncomprising a biarsenical fluorophore, a separately packaged secondcomposition comprising a reducing agent and a thiol competitor, andoptionally a third composition comprising a blocking reagent. Asdescribed hereinabove, in some kit embodiments of the present inventionthe reducing agent and the thiol competitor may be the same agent.

The kits may include a plurality of one or more of the above-describedcompositions, and may further include compositions for effecting celllysis, and compositions that can be diluted to serve as loading orrunning buffers for gel electrophoresis. In some embodiments, the kitfurther includes a separate composition comprising protein standards,such as protein standards whose fluorescence is spectrally-matched tothat of the biarsenical fluorophore. In some kit embodiments, theincluded protein standards may be pre-labeled with the fluorophore, suchas by the biarsenical fluorophore. Alternatively, or in addition, thekit may include protein standards that include a suitable motif, such asthe tetracysteine motif, such that the protein standards can be labeledwith a biarsenical fluorophore, such as by using the compositionsalready included with the kit.

In addition to a composition of the invention, a kit of the inventionmay comprise: one or more sets of instructions; one or moreelectrophoretic media, such a precast gel or compositions and materialsfor preparing gels; one or more antibodies that bind to a protein tag.

As described above, in some embodiments of the methods of the presentinvention, the biarsenical fluorophore can be covalently ornoncovalently attached to a support, such as the supports describedabove. In embodiments in which the support is a plurality of magnetic,paramagnetic, or superparamagnetic beads, the kits of the presentinvention can usefully further comprise apparatus for magneticseparation of the beads within a fluid suspension.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein may be made without departing from thescope of the invention or any embodiment thereof. Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following Examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

EXAMPLES

The examples presented herein provide exemplary instructions for thepractice of some embodiments of the methods of the present invention.

Example 1 Exemplary Results with Tagged Proteins Expressed in E. Coli

Human glucuronidase beta (GUS), human kinase, and chloramphenicolacetyltransferase (CAT) were expressed as tetracysteine-tagged fusionsin BL21 Star™ E. coli cells (Invitrogen Corp., Carlsbad, Calif.).Uninduced control samples, samples 2 hours post induction, and samples 4hours post induction were lysed with BugBuster reagent (Novagen, Inc.,Madison, Wis.) and processed for visualization of the tagged proteins.

Briefly, 5 μL of 4× labeling sample buffer was added to about 15 μLprotein sample.

The 4× labeling sample buffer contains, per 10 ml: (i) 377 μL of 400 mMTBP stock solution (102.6 μL neat Tri-n-butylphosphine (Sigma-Aldrich)to 897.4 μL isopropanol), (ii) 185 μL beta mercaptoethanol(Sigma-Aldrich), and (iii) 9.43 ml 4× sample buffer (containing, per 10ml, glycerol (4.0 g), Tris Base (1.364 g), Tris HCl (1.332 g), LDS (0.8g), EDTA (0.006 g) and Serva Blue G (0.75 ml of 1% solution (SO-BLUE)).

To the protein sample was then added 0.2 μL of Lumio™ Green detectionreagent. An exemplary detection reagent comprises 1 mM biarsenicalfluorophore in a 90% DMSO solution. The sample was heated at 70° C. for10 minutes, then centrifuged for 10 seconds to collect the condensatefrom the lid of the tube.

An aliquot of 2 AL of thiol-alkylating reagent was added and the sampleincubated for five minutes at room temperature. An exemplarythiol-alkylating reagent comprises 1 M maleimide in DMSO solution.

Treated samples were then loaded on a 4-12% Bis-Tris NuPAGE® gel(Invitrogen Corp., Carlsbad, Calif.) and electrophoresed according tostandard protocols with MES running buffer.

Results are shown in FIGS. 5A (fluorescence detection) and 5B (visualdetection after subsequent staining with SimplyBlue™ stain (InvitrogenCorp., Carlsbad, Calif.). Lanes were loaded as follows:

-   Lane 1: 3 μL BenchMark™ fluorescent standard-   Lane 2: 10 μL GUS, uninduced-   Lane 3: 10 μL GUS, 2 hour induction-   Lane 4: 10 μL GUS, 4 hour induction-   Lane 5: 10 μL human kinase, uninduced-   Lane 6: 10 μL human kinase, 2 hour induction-   Lane 7: 10 μL human kinase, 4 hour induction-   Lane 8: 10 μL CAT, uninduced-   Lane 9: 10 μL CAT, 2 hour induction-   Lane 10: 10 μL CAT, 4 hour induction.

Results demonstrate excellent specificity of fluorescence labeling withvery low nonspecific background fluorescence.

Example 2 Exemplary Results with Tagged Proteins Expressed in Vitro

Krev, Jun, CAT and E2F1 were expressed as tetracysteine-tagged fusionsin vitro using the Expressway expression system (Invitrogen Corp.,Carlsbad, Calif.), essentially according to manufacturer's instructions.

Samples were then treated for labeling of the tetracysteine tagessentially as in Example 1, above, and electrophoresed on a 4-12%NUPAGE® gel run with MES running buffer. Results are shown in FIGS. 6A(fluorescence detection) and 6B (visual detection after subsequentstaining with SimplyBlue™ stain (Invitrogen Corp., Carlsbad, Calif.).Lanes were loaded as follows:

-   Lane 1: 5 μL BenchMark fluorescent standard-   Lane 2: 10 μL control (no DNA)-   Lane 3: 10 μL Krev expression-   Lane 4: 10 μL Jun expression-   Lane 5: 10 μL CAT expression-   Lane 6: 10 μL E2F1 expression

Results demonstrate excellent specificity of fluorescence labeling withvery low nonspecific background fluorescence.

Example 3 Exemplary Results with Tagged Proteins Expressed in MammalianCells

Orf6 and Orf7 tetracysteine-tagged proteins were expressed in GripTite™cells, an engineered human 293 embryonic kidney cell line (InvitrogenCorp., Carlsbad, Calif.), according to manufacturer's instructions andstandard protocols. Cell lysates were treated for labeling of thetetracysteine tag essentially as in Example 1, above, andelectrophoresed on a 4-12% NuPAGE® gel run with MES running buffer.Results are shown in FIGS. 7A (fluorescence detection) and 7B (visualdetection after subsequent staining with SimplyBlue™ stain (InvitrogenCorp., Carlsbad, Calif.). Lanes were loaded as follows:

-   Lane 1: 5 μL BenchMark™ fluorescent standard-   Lane 2: 10 μL mock transfected control-   Lane 3: 10 μL Orf6 lysate-   Lane 4: 10 μL Orf7 lysate

Results demonstrate excellent specificity of fluorescence labeling withvery low nonspecific background fluorescence.

Example 4 Real-Time Monitoring of Tetracysteine-Tagged Protein Synthesis

Reducing agent, thiol competitor, and biarsenical fluorophore are addedto the Expressway Plus™ Lumio™ cell-free in vitrotranscription-translation extract (Invitrogen Corp., Carlsbad, Calif.).Protein production is observed in real-time by measuring directly from amicrotiter plate using a 96 well plate reader. The excitation wavelengthis set at 500 nm, while emission is monitored at 535 nm. Readings aretaken at 10 minute intervals over a two hour incubation period,providing data analogous to those shown in FIG. 9.

All patents, patent publications, and published references cited hereinare incorporated herein by reference in their entireties as ifspecifically and individually incorporated.

Although a number of embodiments and features are described herein, itwill be understood by those skilled in the art that modification andvariations of the described embodiments and features may be made withoutdeparting from either the spirit of the invention or the scope of theappended claims. All publications and patents cited herein areincorporated by reference in their entireties.

1. A method of labeling a tetracysteine-tagged protein species presentin a protein sample with a biarsenical fluorophore, the methodcomprising: treating the proteins in said sample in the presence of acomposition comprising a thiol competitor and a biarsenical fluorophore.2. The method of claim 1, wherein said composition further comprises areducing agent.
 3. The method of claim 2, wherein said reducing agent istributylphosphine (TBP).
 4. The method of claim 1, wherein said thiolcompetitor is a monothiol.
 5. The method of claim 4, wherein saidmonothiol is beta-mercaptoethanol (BME) and the BME is at aconcentration greater than about 1 mM.
 6. The method of claim 5, whereinsaid BME is at a concentration of at least about 5 mM.
 7. The method ofclaim 6, wherein said BME is at a concentration of at least about 60 mM.8. The method of claim 1, wherein said thiol competitor is a dithiolwith concentration of at least 100 μM.
 9. (canceled)
 10. The method ofclaim 2, wherein said reducing agent is at a concentration of at leastabout 1 mM. 11-12. (canceled)
 13. The method of claim 1, wherein saidbiarsenical fluorophore is a biarsenical derivative of fluorescein. 14.The method of claim 13, wherein said biarsenical fluorophore is4′-5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(2,2-ethanedithiol)2. 15.(canceled)
 16. The method of claim 2, wherein said biarsenicalfluorophore is added to said protein sample after said thiol competitorand said reducing agent are added. 17-19. (canceled)
 20. The method ofclaim 16, wherein said treating step is at a temperature of about 70° C.21-24. (canceled)
 25. The method of claim 1, wherein said treating isfor a period of no more than about 10 minutes.
 26. The method of claim1, further comprising a later step of: resolving the proteins in saidsample by gel electrophoresis.
 27. The method of claim 26, comprising ayet further step of detecting said tetracysteine-tagged protein amongsaid resolved proteins in said gel.
 28. The method of claim 1, furthercomprising a later step of: treating said protein sample with athiol-blocking reagent.
 29. The method of claim 28, wherein saidthiol-blocking reagent is selected from the group consisting of:alkylhalides, iodoacetamide, iodoacetic acid, dithiobis(2-nitrobenzoicacid) (DTNB), dithiobis(5-nitropyridine), maleimide andethylenemaleimide.
 30. (canceled)
 31. The method of claim 29, whereinsaid thiol-blocking reagent is maleimide. 32-39. (canceled)
 40. Themethod of claim 1, wherein said biarsenical fluorophore is bonded to asurface of a support.
 41. The method of claim 40, wherein said supportcomprises a plurality of beads. 42-47. (canceled)
 48. A composition forimproving specificity of labeling of tetracysteine-tagged proteins witha biarsenical fluorophore, comprising: a biarsenical fluorophore; and athiol competitor.
 49. The composition of claim 48 further comprising areducing agent, wherein said reducing agent is tributylphosphine (TBP).50. The composition of claim 48, wherein said thiol competitor is amono- or dithiol.
 51. The composition of claim 50, wherein said thiolcompetitor is beta-mercaptoethanol (BME).
 52. A kit for labelingtetracysteine-tagged proteins with a biarsenical fluorophore,comprising: a first composition comprising a thiol competitor.
 53. Thekit of claim 52, wherein said first composition further comprises abiarsenical fluorophore.
 54. The kit of claim 52, further comprising asecond composition comprising a biarsenical fluorophore.
 55. The kit ofclaim 52, further comprising: a composition comprising a blockingreagent.
 56. The kit of claim 52, wherein said thiol competitor is amono- or dithiol.
 57. The kit of claim 56, wherein said thiol competitoris beta-mercaptoethanol (BME).
 58. The kit of claim 52, wherein saidfirst composition further comprises a reducing agent, wherein saidreducing agent is a phosphine.
 59. The kit of claim 58, wherein saidphosphine is tributylphosphine (TBP).
 60. The kit of claim 52, whereinsaid biarsenical fluorophore is a biarsenical fluorescein derivative.61. (canceled)
 62. The kit of claim 55, wherein said blocking reagent isselected from maleimide and iodoacetamide.
 63. The kit of claim 62,where said blocking reagent is maleimide. 64-67. (canceled)
 68. Thecomposition of claim 48 further comprising at least one buffer, an ionicdetergent, and a marker dye.
 69. The composition of claim 68, whereinthe buffer comprises Tris and EDTA; and the ionic detergent is adodecylsulfate salt.